Transgenic rat models will be developed by genetic modification and transplantation of male germ line stem cells. Rats are widely used models of basic biology, and human physiology and disease, but the study of genes and their functions in the rat lags far behind progress in the mouse. While production and analysis of gain-of-function (transgenic, knock-in) and loss-of-function (gene targeted knockout) mutant mice have become routine and provide the most direct way to correlate specific genes with phenotypes, transgenic production in rats and other species is inefficient, with knockout technology unavailable. Development of rat genomic resources (the Rat Expressed Sequence Tag [EST] program and the Rat Genome program), combined with decades of phenotypic and physiological data, provide unprecedented opportunities for establishing rat models of mammalian biology. Spermatogonial stem cells constitute a unique vehicle for modifying the germ line, and it has been established that genetic manipulation and transplantation of this male germ line stem cell provides an alternative means for the production of transgenic mice. Since the spermatogenic process is well conserved across mammalian species, it is hypothesized that this new technology will be readily translated to rats. Furthermore, development of conditions for maintaining or expanding spermatogonial stem cells in culture will improve efficiency and expand the repertoire of genetic approaches that can be implemented. This technology will be developed in the rat because the rat is an established model for human physiology and disease, but the results should have broad implications for the development of transgenesis in other species. The recently described spermatogonial transplantation technique will be used to address and enable the following specific aims: 1) produce donor rat testis cell populations enriched for spermatogonial stem cells and develop appropriate rat recipient models; 2) introduce genetic modifications into donor rat spermatogonial stem cells and recover the new genotype in progeny of recipient rats; 3) develop conditions for maintaining and expanding rat spermatogonial stem cells in culture to increase their accessibility for genetic modification; and 4) immortalize rat spermatogonial stem cells by introducing the gene encoding telomerase reverse transcriptase (TERT) to establish the foundation for generating targeted genetic changes.
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